PROJECT SUMMARY Free cholesterol is an essential structural component required for integrity and fluidity of membrane bilayers important for cell signaling and regulation of lipid metabolism. Free cholesterol is also the precursor for the synthesis of steroid hormones and bile acids. The balance between free cholesterol and its esterified storage form is tightly regulated by all cells in the body, and is crucial for preventing hypercholesterolemia and associated disorders. Until recently, it was believed that the translocator protein (TSPO) present in the outer mitochondrial membrane was a transporter for cholesterol to enter the mitochondria for steroid hormone production. We overturned this assumption in recent work demonstrating that TSPO is not involved in this process. This void has refocused attention on elucidating the role of TSPO action. This is of extreme importance because upregulation of TSPO is seen in multiple human pathologies, and there are currently 24 human clinical trials targeting TSPO for therapeutic or diagnostic purposes. By reexamining the relationship between TSPO and cholesterol using different Tspo gene deleted models we have identified a novel link between TSPO and cholesterol esterification. The objective of this application is to elucidate the precise role of TSPO: (Aim 1) Define the TSPO-mediated mechanism in cholesterol esterification, and (Aim 2) Understand the in vivo changes to lipid homeostasis in Tspo knockout (Tspo-/-) mice. The experimental plans for this project extend from molecular characterization of TSPO to studying its functional relevance using in vivo metabolism models. Using in vitro models, we investigate the importance of cholesterol binding and protein interactions to TSPO by evaluating specific amino acid mutations to putative binding sites; we also perform metabolomics to evaluate cellular responses and determine functional integration with known pathways of cholesterol regulation in cells. Using in vivo models, we examine TSPO effects on lipid metabolism in the liver and white adipose tissue and the associated impact on plasma lipids. Results from these studies are poised to offer novel insights into lipid regulation by TSPO that might explain specific pathological dysfunctions observed in atherosclerosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). In addition, knowledge of TSPO function will be highly relevant to describing the basis of its involvement in pathologies and defining the principles underlying pharmacological targeting of TSPO for advancing human medicine.